ABSTRACT ? PROJECT 3 It is estimated that in the next 20 years, the number of individuals in the United States over the age of 65 will double, reaching more than 70 million individuals, many with multiple age-related conditions. Thus there is a tremendous need to identify therapeutic strategies to extend healthspan. Current approaches include the testing of candidate drugs in worms and mice by the CITP and ITP and screening in cell culture for compounds that affect specific activities related to aging such as mitochondrial function. Indeed, research with model organisms has identified multiple promising genes and pathways that potentially can be manipulated in humans to delay aging and its pathological consequences through pharmacological means. However, similar genetic approaches can be applied directly to humans, using genetic variation in human populations that is linked to phenotypes of healthy aging or disease-free longevity. These natural occurring genetic variants in humans that affect longevity and healthspan offer an ideal starting point for taking interventions that promote healthspan. The best examples of such ?natural longevity mutants? are human centenarians and super- centenarians, many of whom managed to ward off the diseases that normally begin to plague humans at middle age. In this U19 application, we propose to apply this genome-to-drug approach using sequencing analysis of centenarians to the development of drugs able to extend human healthspan. Project 1 has identified rare variants in human centenarians associated with extended healthspan including IGF-1R, SIRT6, FOXO3A, ATM and components of the NF??? pathway, RelA/p65, NFKB1/p50 and in particular, NFKB1a (I?B?). These rare variants and miRNAs are being validated in mouse models of aging in Project 2. We previously have developed cell-based fluorescent assays for identifying novel compounds able to act as senotherapeutics that can extend healthspan in mouse models of aging. Here we will develop assays for screening for drugs, including a library of clinically used compounds, which mimic these identified genetic variants or associated pathways. Indeed, we already have developed novel and effective small molecule inhibitors of the canonical NF-?B pathway able to extend healthspan in mouse models. In addition, we have demonstrated the ability to develop small molecules targeting specific miRNAs, for example, TGP-96, which targets and inhibits miR-96, upregulating FOXO3a and FOXO1 and decreasing cellular senescence. Thus we also will develop novel small molecules targeting miRNAs that preferentially go down in centenarians with extended longevity. The specific Aims of the proposal are: 1) To develop, test and optimize assays for screening for compounds able to mimic the effect of rare variants in IGF-1R, SIRT6, FOXO3A, ATM and the IKK/I?B?/NF-?B pathway; 2) To develop small molecule inhibitors of the miRNAs identified as being downregulated in centenarians; and 3) To test the ability of these compounds to extend healthspan in mouse models of aging in collaboration with Project 2.